In chromosome DNA of mammalian cells, cytosine in CpG islands composed of a CG dinucleotide sequence is known to be methylated or demethylated at its position 5. Methylation and demethylation of cytosine in CpG islands regulates the transcriptional regulation mechanism of genes. Typically, in the promoter region of a gene, a region containing many CpG islands is present, and the presence and absence of methylation of cytosine in the promoter region of a gene functions as an on-off switch for transcription of the gene. Although methylation of cytosine has long been studied, the details of cytosine demethylation have remained unknown for a long time. However, Non-patent Literature 1 has recently revealed that deamination of 5-methylcytosine or oxidation of 5-methylcytosine into 5-hydroxymethylcytosine causes the cytosine to protrude from the double helical structure, and thus causes the N-glycosidic site to undergo hydrolysis by an enzyme, thereby generating an abasic site (apyrimidinic site: AP site) opposite guanine (FIG. 1).
Cytosine demethylation is regulated by enzymes. However, the bases of the genomic DNA in mammalian cells are exposed to chemical substances, ultraviolet ray or X-ray irradiation, or oxidation stress, and 50,000 to 200,000 bases per day are eliminated at random, creating abasic sites. It is known that, as a result, a deoxyribose having an acetal structure generated by the elimination of any of four bases of DNA opens the ring by an equilibrium reaction and transforms into aldehyde (FIG. 2). Because the damage caused by the elimination of a base of DNA can be deeply involved in the transcription or translation of a gene, the analysis of the damage is important. At present, biotinylated hydroxylamine compounds disclosed in prior art (Non-patent Literature 2, Patent Literature 1) are commercially available. The reagents specifically bind to an abasic lesion and detect the coloration or luminescence caused by an enzyme using biotinylation and the specific bond with an avidin-enzyme conjugate. This detection technique is widely used, and exhibits high sensitivity and excellent quantification. However, the reagents are intended for measuring the average amount of abasic sites for the four bases of DNA, and cannot identify the position of the abasic sites in DNA or the type of corresponding bases in the complementary strand.
Formaldehyde, glyoxal, and a kethoxal compound, which is a derivative of glyoxal, have been long known to react with the amino group at position 2 of guanine in a single-stranded RNA or DNA (Non-patent Literature 3). In particular, glyoxal and its derivative, kethoxal, are known to react with guanine to form three ring structures. The reaction yield has recently been measured by mass spectrometry, which revealed that the reaction yield varies widely depending on the position of the guanine base in DNA, ranging from 9 to 89% (Non-patent Literature 4). However, even if 9 to 89% of guanine in a single-stranded DNA is modified, the DNA with 10% or more of unreacted guanine would sufficiently serve as a template for polymerase chain reaction, having little influence on PCR, which amplifies the DNA fragment by 2 to the power of n.
Endonucleases, which act on their substrate, abasic sites of DNA, are classified into two types: endonuclease IV and APEI nuclease, which are derived from E. coli, and DNA endonuclease III, which is derived from E. coli Nth or the like. The former recognizes an abasic site in a DNA double helix and cleaves it off between 3′ OH and 5′ phosphoric acid. The latter enzyme forms a Schiff base with an abasic site of a deoxyribose, and 5′ phosphoric acid is released from the ribose by beta-elimination in the Schiff base. When a base opposite the abasic site generated in a double-stranded DNA has been chemically modified, the amino acid residue at the active center of the endonuclease is expected to no longer bind to DNA.